1 . Field of the Invention
This invention relates to a diagnostic kit and a method for determining the concentration of sucrose in a sample of a physiological fluid. The kit and method are useful in connection with a method for the non-invasive detection of gastric damage.
2. Related Background Art
Stomach ulcers can pose a serious health threat as, in many instances, ulcers are asymptomatic. Since stomach ulcers can develop and be present without any symptoms, the damage brought about by ulcers to the stomach and the bleeding associated with such ulcers can be a serious, and possibly fatal health risk.
Traditional methods for detecting gastric ulcers include endoscopy, barium meal followed by x-rays, and radiolabeled detecting agents. Endoscopy causes patient discomfort, requires anesthesia, and must generally be performed in a clinic or a hospital. X-rays and radiolabeled detecting agents have the common disadvantage of exposing the patient to radiation. In addition, all of these procedures require a skilled evaluation of the results in order to properly diagnose the patient's condition.
A method for detection of gastric epithelial damage, particularly ulcers and lesions in the stomach, using non-invasive, non-radioactive and non-x-ray techniques or procedures is disclosed in U.S. Pat. No. 5,620,899. The method of this reference employs a disaccharide which can be orally administered to a patient, which does not transport across cell membranes, which is metabolized within the small intestine to its monosaccharide components, and which is not broken down elsewhere in the body. Damage to the gastric epithelium will allow the disaccharide to enter the blood without being metabolized. Hence, the disaccharide will appear in the blood or urine to an extent that can be correlated with the extent of gastric epithelial damage. Typically, the disaccharide is administered to a patient, followed by collection of blood or urine, which is assayed for the disaccharide. The use of sucrose in particular as a diagnostic marker in detection of gastric epithelial damage is described in U.S. patent application Ser. No. 08/456,203.
The methodology utilized to evaluate the level of sucrose in the blood or urine in conjunction with the performance of the above-described gastric assay must be useful for analyzing physiological fluids. A hexokinase/glucose-6-phosphate method has been suggested for analysis of glucose in serum, plasma, or urine. United States Department of Health, Education and Welfare, Food and Drug Administration. In Vitro Diagnostic Products for Human Use, Proposed Establishment of Product Class Standard for Detection or Measurement of Glucose, Fed. Regist. Vol. 39, No. 126, 24146 (1974). There is, however, no suggestion of measuring sucrose in physiological fluids.
A method is described in S. Sekin, Tobacco International, Vol. 181, 27-27 (1979) for determination of sucrose in tobacco. This method converts glucose in an aliquot of a tobacco sample to 6-phosphogluconate, with concomitant production of NADH, by addition of ATP, hexokinase, glucose-6-phosphate dehydrogenase and NAD. An absorbance measurement at 340 nm is used to quantify the NADH, and hence the endogenous glucose. Another aliquot is treated with invertase, ATP, hexokinase, glucose-6-phosphate dehydrogenase and NAD. The invertase cleaves the sucrose to glucose and fructose. The glucose produced from cleavage, along with the glucose initially present in the sample, is converted to 6-phosphogluconate, producing NADH. An absorbance measurement at 340 nm quantifies NADH, and hence total glucose. The difference between total glucose and endogenous glucose gives the molar amount of sucrose present. However, there is no suggestion that this method would be compatible with analyzing sucrose in complex physiological fluids. In addition, the reagents used for the analysis are non-stabilized solutions which cannot be stored or transported for long periods of time.
A method suitable for determination of sucrose in physiological fluids would be highly desirable, as would a diagnostic kit containing the necessary reagents preformulated for use in such a method.